Our previous work demonstrated that HIV-1 infection progressively reduces TCR/CD3 expression due to a defect in CD3g gene transcripts. We further found that knocking down expression of the viral tat and/or nef genes was correlated with CD3g transcript and TCR/CD3 surface receptor levels on HIV-1 infected cells. This study was undertaken to investigate the direct effect of HIV-1 Tat expression on the TCR/CD3 machinery. Progressive downregulation from TCR/CD3hi to TCR/CD3lo to TCR/CD3 was observed on Tat expressing cells in a manner that emulated HIV-1 infection, with a lack of CD3g transcripts again responsible for the defect. When Tat cell cultures containing a mixture of TCR/CD3 surface densities were separated into TCR/CD3hi and TCR/CD3lo/ populations, they quickly reverted to a mixed CD3 phenotype. Thus, the progression TCR/CD3hi to TCR/CD3lo to TCR/CD3 is an active, reversible process with receptor levels fluctuating in response to intracellular dynamics. Examination of tat mutants found that the regions involved in Tat-mediated transactivation and TAR binding are required for TCR/CD3 downregulation while the lysine at position 28 and Tat exon 2 are dispensable. Global gene expression, assessed in association with TCR/CD3 downregulation in HIV-1 infected and Tat expressing cells, detected broad suppression of TCR/CD3 signaling, co-stimulation and negative regulatory genes along with target transcription factors, ligands and receptors. A significant subset of the genes altered in HIV-1 infected cells was specifically targeted by Tat in association with TCR/CD3 loss. Our finding that Tat negatively regulates many facets of the TCR/CD3 machinery has important implications for disease pathogenesis.